DOT1L is the lysine methyltransferase responsible for histone H3 lysine 79 (H3K79) methylation and plays a crucial role in leukemia progression. Furthermore, DOT1L has biological functions that are independent of its methyltransferase activity. Therefore, targeting and degrading DOT1L with PROteolysis TArgeting Chimeras (PROTACs) could represent a promising therapeutic strategy. Here, we report the discovery of the first-in-class DOT1L PROTAC degrader, compound 13 (MS2133), which potently induces DOT1L degradation in a concentration- and time-dependent manner, without affecting DOT1L mRNA expression. The DOT1L degradation induced by 13 requires binding to the E3 ligase von Hippel-Lindau (VHL) and DOT1L and occurs through the ubiquitin-proteasome system. 13 is selective for DOT1L over other methyltransferases and effectively inhibits the growth of mixed lineage leukemia-rearranged (MLL-r) leukemia cells while having no toxicity on normal cells. Overall, 13 is a valuable chemical biology tool for further studying functions of DOT1L and a potential therapeutic for DOT1L-dependent cancers.

Genomes adapt dynamically to alterations in the signaling milieu, including inflammation that transiently or permanently disrupts genome function. Here, we elucidate how a progenitor cell genome senses and responds to inflammation when the developmental and transcriptional regulator GATA2 is limiting, which causes bone marrow failure in humans and mice and predisposes to leukemia in humans. GATA2low murine progenitors are hypersensitive to inflammatory mediators. We discovered that the hematopoietic transcription factor PU.1 conferred transcriptional activation in GATA2low progenitors in response to Interferon-γ and Toll-Like Receptor 1/2 agonists. In a locus-specific manner, inflammation reconfigured genome activity by promoting PU.1 recruitment to chromatin or tuning activity of PU.1-preoccupied chromatin. The recruitment mechanism disproportionately required IKKβ activity. Inflammation-activated genes were enriched in motifs for RUNX factors that cooperate with GATA factors. Contrasting with the GATA2-RUNX1 cooperativity paradigm, GATA2 suppressed and RUNX1 promoted PU.1 mechanisms to endow the progenitor genome with inflammation-sensing capacity.

Emberger syndrome (ES), an autosomal dominant disorder characterized by congenital deafness, primary lymphedema, and predisposition to myeloid malignancies, is caused by mutations in the GATA2 gene. Although primary lymphedema is an important hallmark of ES, the pathophysiology remains unclear due to the lack of a suitable experimental model. In this study, we isolated induced pluripotent stem cells (iPSCs) from two patients with ES (i.e., ES-iPSCs) and analyzed their in vitro lymphatic differentiation potential via the mesodermal progenitor stage. KDR+ CD34+ early mesodermal progenitors generated from either ES-iPSCs or wild-type iPSCs during a 6-days serum- and feeder-free culture supplemented with bone morphogenetic protein 4 and vascular endothelial growth factor (VEGF) had almost equivalent developmental potential. However, upon co-culture with OP9 stromal cells, KDR+ CD34+ cells derived from ES-iPSCs developed into CD31+ lymphatic vessel endothelial hyaluronan receptor-1+ VEGF receptor 3+ lymphatic endothelial cells less efficiently than KDR+ CD34+ cells derived from wild-type iPSCs. Thus, patient-derived iPSCs recapitulate impairments at an early stage of lymphangiogenesis, making them a useful experimental tool for dissecting the pathophysiology of primary lymphedema in ES and developing potential therapeutic approaches.

Stemness-associated cell states are linked to chemotherapy resistance in acute myeloid leukemia (AML). We uncovered a direct mechanistic link between expression of the stem cell transcription factor GATA2 and drug resistance. The GATA-binding protein 2 (GATA2) plays a central role in blood stem cell generation and maintenance. We find substantial intrapatient and interpatient variability in GATA2 expression across samples from patients with AML. GATA2 expression varies by molecular subtype and has been linked to outcome. In a murine model, KMT2A-MLL3-driven AML originating from a stem cell or immature progenitor cell population has higher Gata2 expression and is more resistant to the standard AML chemotherapy agent doxorubicin. Deletion of Gata2 resulted in a more robust induction of p53 after exposure to doxorubicin. Chromatin immunoprecipitation sequencing, RNA sequencing, and functional studies revealed that GATA2 regulates the expression of RASSF4, a modulator of the p53 inhibitor MDM2 (mouse double minute 2). GATA2 and RASSF4 are anticorrelated in human cell lines and in bulk and single-cell expression data sets from patients with AML. Knockdown of Rassf4 in Gata2-low cells resulted in doxorubicin or nutlin-3 resistance. Conversely, overexpression of Rassf4 results in sensitization of cells expressing high levels of Gata2. Finally, doxorubicin and nutlin-3 are synergistic in Gata2-high murine AML and in samples from patients with AML. We discovered a previously unappreciated role for GATA2 in dampening p53-mediated apoptosis via transcriptional regulation of RASSF4, a modulator of MDM2. This role for GATA2 directly links the expression of a stemness-associated transcription factor to chemotherapy resistance.

Androgens drive the morphogenesis and differentiation of the Wolffian duct (WD) into the epididymis, an essential organ for male reproduction, by binding to the androgen receptor (AR). However, it remains unclear whether other transcriptional programs operate beyond the central androgen/AR signaling in promoting WD development. We discovered that mesenchyme-specific deletion of the transcription factor Gata2 resulted in defective epididymal coiling in the corpus and caudal regions. The defective coiling in the absence of mesenchymal Gata2 did not result from androgen signaling deficiency, as there were no abnormalities in testicular morphology, androgen production, or AR/Ar expression, and dihydrotestosterone supplementation did not restore epididymal coiling in cultured WDs. Instead, Gata2 deletion reduced the expression of the mesenchyme-derived factor Inhba and epithelial proliferation, both of which play critical roles in epididymal coiling. The epididymal defect persisted into adulthood, with the uncoiled corpus and caudal epididymis exhibiting abnormal epithelial morphology and lumen environments, resulting in an unfavorable environment for sperm storage. Our results demonstrate the androgen-independent role of mesenchymal GATA2 in promoting epididymal development through Inhba induction and highlight the importance of proper fetal development in male reproduction.

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) represent complex hematopoietic malignancies characterized by ineffective hematopoiesis and dysregulated myeloid differentiation. Recent research has underscored the critical role of aberrant STAT signaling pathways, particularly involving STAT3 and STAT5, in the pathogenesis of these disorders. Aberrant activation of STAT proteins has been implicated as a mediator of oncogenesis in several malignancies. In this review, we discuss the role of STAT proteins in both regulated and dysregulated hematopoiesis, the consequences of dysregulation in acute myeloid leukemia and myelodysplastic syndromes, therapeutic strategies, and recent advancements in STAT-targeted therapy. By integrating findings from recent preclinical and clinical studies, this review provides insights into the evolving landscape of STAT-targeted therapies, highlighting the promise of these approaches in enhancing treatment efficacy and improving patient outcomes in high-risk hematologic malignancies.

GATA2 deficiency is an autosomal dominant germline disorder of immune dysfunction and bone marrow failure with a high propensity for leukemic transformation. While sequencing studies have identified several secondary mutations thought to contribute to malignancy, the mechanisms of disease progression have been difficult to identify due to a lack of disease-specific experimental models. Here, we describe a murine model of one of the most common GATA2 mutations associated with leukemic progression in GATA2 deficiency, Gata2R396Q/+. While mutant mice exhibit mild defects in peripheral blood, they display significant hematopoietic abnormalities in the bone marrow, including a reduction in hematopoietic stem cell (HSC) function and intrinsic biases toward specific stem cell subsets that differ from previous models of GATA2 loss. Supporting this observation, single-cell RNA sequencing of hematopoietic progenitors revealed a loss of stemness, myeloid-bias, and indications of accelerated aging. Importantly, we show that Gata2R396Q/+ exerts effects early in hematopoietic development, as mutant mice generate fewer HSCs in the aorta gonad mesonephros, and fetal liver HSCs have reduced function. This reduced and altered pool of HSCs could be potential contributors to leukemic transformation in patients, and our model provides a useful tool to study the mechanisms of malignant transformation in GATA2 deficiency.

Patients with chronic, indolent myeloproliferative neoplasms (MPNs) are at risk for transformation to highly lethal leukemia, although targetable mechanisms driving progression remain elusive. We discovered that the High Mobility Group A1 (HMGA1) gene is up-regulated with MPN progression in patients and required for evolution into myelofibrosis (MF) or acute myeloid leukemia (AML) in preclinical models. HMGA1 encodes the HMGA1 epigenetic regulators that modulate the chromatin state during embryogenesis and tissue regeneration. While HMGA1 is silenced in most differentiated cells, it becomes aberrantly re-expressed in JAK2 mutant (JAK2-V617F) MPN, with the highest levels after transformation to secondary MF or AML. Here, we review recent work highlighting HMGA1 function in MPN progression. Though underlying mechanisms continue to emerge, increasing evidence suggests that HMGA1 functions as a "chromatin key" required to "unlock" regions of the genome involved in clonal expansion and progression in MPN. Together, these findings illuminate HMGA1 as a driver of MPN progression and a promising therapeutic target.

Runs of Homozygosity (ROH) regions are characterized by homozygous genotypes inherited from a common ancestor, often arising from positive selection for adaptive traits. These homozygous regions may arise due to inbreeding, selective breeding, or demographic events like population bottlenecks. Transcription factor binding sites (TFBS) are short, specific DNA sequences where transcription factors bind to regulate the expression of nearby genes. These sites are essential for controlling biological processes such as development, metabolism, and immune response. TFBS act as key regulatory elements, and their variations can influence gene activity, contributing to phenotypic differences and adaptation. ROH often encompass regulatory elements, including TFBS, suggesting a functional connection between these genomic features. This study investigates TFBS within ROH regions in 297 animals of six cattle breeds: Gir (48), Tharparkar (72), Vrindavani (72), Frieswal (14), Holstein Friesian (63), and Jersey (28). Utilizing genotyped data of these animals, we identified genomic regions enriched with ROH. We focused on the central 10 kb regions of 50 ROH regions common across all breeds. Within these regions, 450 motifs were examined, identifying 168 transcription factors potentially binding to these regions. The results emphasize the role of TFBS in gene regulation and adaptive processes. By linking ROH patterns to regulatory elements, this study enhances our understanding of the genetic architecture underlying phenotypic traits and their adaptation to environmental pressures. These findings provide insights into the molecular mechanisms influencing genetic variation in cattle populations.

GATA2 germline mutations lead to a syndrome characterized by immunodeficiency, vascular disorders and myeloid malignancies. To elucidate how these mutations affect hematopoietic homeostasis, we created a knock-in mouse model expressing the recurrent Gata2 R396Q missense mutation. Employing molecular and functional approaches, we investigated the mutation's impact on hematopoiesis, revealing significant alterations in the hematopoietic stem and progenitor (HSPC) compartment in young age. These include increased LT-HSC numbers, reduced self-renewal potential, and impaired response to acute inflammatory stimuli. The mature HSPC compartment was primarily affected at the CMP sub-population level. In the mutant LT-HSC population, we identified an aberrant subpopulation strongly expressing CD150, resembling aging, but occurring prematurely. This population showed hyporesponsiveness, accumulated over time, and exhibited allele-specific expression (ASE) favoring the mutated Gata2 allele, also observed in GATA2 mutated patients. Our findings reveal the detrimental impact of a Gata2 recurrent missense mutation on the HSC compartment contributing to its functional decline. Defects in the CMP mature compartment, along with the inflammatory molecular signature, explain the loss of heterogeneity in HPC compartment observed in patients. Finally, our study provides a valuable model that recapitulates the ASE-related pathology observed in GATA2 deficiency, shedding light on the mechanisms contributing to the disease's natural progression.

Myeloid neoplasms with and without preexisting platelet disorders frequently develop in association with an underlying germline predisposition. Germline alterations affecting ANKRD26, CEBPA, DDX41, ETV6, and RUNX1 are associated with nonsyndromic predisposition to the development of myeloid neoplasms including acute myeloid leukemia and myelodysplastic syndrome. However, germline predisposition to myeloid neoplasms is also associated with a wide range of other syndromes, including SAMD9/9L associated predisposition, GATA2 deficiency, RASopathies, ribosomopathies, telomere biology disorders, Fanconi anemia, severe congenital neutropenia, Down syndrome, and others. In the fifth edition of the World Health Organization (WHO) series on the classification of tumors of hematopoietic and lymphoid tissues, myeloid neoplasms associated with germline predisposition have been recognized as a separate entity. Here, we review several disorders from this WHO entity as well as other related conditions with an emphasis on the molecular pathogenesis of disease and accompanying somatic alterations. Finally, we provide an overview of establishing the molecular diagnosis of these germline genetic conditions and general recommendations for screening and management of the associated hematologic conditions.

Most blood cells derive from hematopoietic stem cells (HSCs), originating from endothelial cells. The induction of HSCs from endothelial cells occurs during mid-gestation, and research has revealed multiple steps in this induction process. Hemogenic endothelial cells emerge within the endothelium, transition to hematopoietic cells (pre-HSCs), and subsequently mature into functional HSCs. Reports indicate transcription factors and external signals are involved in these processes. In this review, we discuss the timing and role of these transcription factors and summarize the external signals that have demonstrated efficacy in an in vitro culture. A precise understanding of the signals at each step is expected to advance the development of methods for inducing HSCs from pluripotent stem cells.

HBO1, also known as KAT7 or MYST2, is a crucial histone acetyltransferase with diverse cellular functions. It typically forms complexes with protein subunits or cofactors such as MEAF6, ING4, or ING5, and JADE1/2/3 or BRPF1/2/3, where the BRPF or JADE proteins serve as the scaffold targeting histone H3 or H4, respectively. The histone acetylation mediated by HBO1 plays significant roles in DNA replication and gene expression regulation. Additionally, HBO1 catalyzes the modification of proteins through acylation with propionyl, butyryl, crotonyl, benzoyl, and acetoacetyl groups. HBO1 undergoes ubiquitination and degradation by two types of ubiquitin complexes and can also act as an E3 ubiquitin ligase for the estrogen receptor α (ERα). Moreover, HBO1 participates in the expansion of medullary thymic epithelial cells (mTECs) and regulates the expression of peripheral tissue genes (PTGs) mediated by autoimmune regulator (AIRE), thus inducing immune tolerance. Furthermore, HBO1 influences the renewal of hematopoietic stem cells and the development of neural stem cells significantly. Importantly, the overexpression of HBO1 in various cancers suggests its carcinogenic role and potential as a therapeutic target. This review summarizes recent advancements in understanding HBO1's involvement in acylation modification, DNA replication, ubiquitination, immunity, and stem cell renewal.

The GATA gene family encodes highly conserved zinc-finger transcription factors that facilitate the development and function of multiple organ systems including the uterus. In the endometrium, GATA2 functions in a positive autoregulatory loop with the progesterone receptor (PGR) and colocalizes with PGR on chromatin to promote PGR transcriptional programs. GATA2 also has PGR-independent functions that maintain endometrial cell identity, and GATA2 transcripts reportedly are down-regulated in endometrial disorders including endometriosis. This event is accompanied by a reciprocal increase in GATA6. Here, we applied custom anti-GATA2 monoclonal antibodies and performed GATA2 immunohistochemistry (IHC) on patient endometrial tissues corresponding to proliferative, secretory, inactive, and hormone-treated endometrium, as well as endometriosis and endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia (EAH/EIN). We also performed IHC for the estrogen receptor, PGR, and GATA6 in relevant groups. The results reveal a tight correlation between GATA2 and PGR expression in the glandular and stromal cells of benign endometrium. GATA2 expression is markedly reduced in stromal but not glandular cells in endometriosis and EAH/EIN. This reduction in GATA2 expression does not lead to a detectable increase in GATA6 expression in endometriosis. Although average glandular GATA2 expression was preserved in endometriosis and EAH/EIN cases, its expression was decoupled from PGR, implying that alternative pathways regulate GATA2 levels in these disorders. Our findings indicate that GATA2 dysregulation is a feature of endometriosis and EAH/EIN, and support a model whereby loss of stromal GATA2 in these disorders contributes to their progesterone insensitivity.

Most gene functions have been discovered through phenotypic observations under loss of function experiments that lack temporal control. However, cell signaling relies on limited transcriptional effectors, having to be re-used temporally and spatially within the organism. Despite that, the dynamic nature of signaling pathways have been overlooked due to the difficulty on their assessment, resulting in important bottlenecks. Here, we have utilized the rapid and synchronized developmental transitions occurring within the zebrafish embryo, in conjunction with custom NF-kB reporter embryos driving destabilized fluorophores that report signaling dynamics in real time. We reveal that NF-kB signaling works as a clock that controls the developmental progression of hematopoietic stem and progenitor cells (HSPCs) by two p65 activity waves that inhibit cell cycle. Temporal disruption of each wave results in contrasting phenotypic outcomes: loss of HSPCs due to impaired specification versus proliferative expansion and failure to delaminate from their niche. We also show functional conservation during human hematopoietic development using iPSC models. Our work identifies p65 as a previously unrecognized contributor to cell cycle regulation, revealing why and when pro-inflammatory signaling is required during HSPC development. It highlights the importance of considering and leveraging cell signaling as a temporally dynamic entity.

Transcriptional mechanisms establish and maintain complex genetic and protein networks to control cell state transitions. The hematopoietic transcription factor GATA1 is a master regulator of erythropoiesis and megakaryopoiesis, and human GATA1 genetic variants cause anemia and megakaryoblastic leukemia. Multiomic analyses revealed that GATA1 controls expression of transporters and metabolic enzymes that dictate intracellular levels of endogenous small molecules, including heme, metal ions, and sphingolipids. Besides its canonical function as a hemoglobin component, heme facilitates or antagonizes GATA1 function to regulate erythropoiesis via mechanisms dependent or independent of the heme-binding transcription factor BTB domain and CNC homology 1 (BACH1). GATA1 regulates the expression of genes encoding heme biosynthetic enzymes and BACH1. GATA1 maintains homeostasis of bioactive ceramides during erythroid differentiation by regulating genes encoding sphingolipid metabolic enzymes. Disrupting ceramide homeostasis impairs critical cytokine signaling and is detrimental to erythroid cells. During erythroid maturation, GATA1 induces a zinc transporter switch that favors export versus import, thus dictating the intracellular zinc level, erythroblast survival, and differentiation. In aggregate, these studies support an emerging paradigm in which GATA factor-dependent transcriptional mechanisms control the intracellular levels of endogenous small molecules and small molecule-dependent feedback loops that serve as vital effectors of transcription factor activity, genome function, and cell state transitions.

Defining the mechanisms that regulate stem cell maintenance, proliferation, and differentiation is critical for identifying therapies for improving stem cell function under stress. Here, we have identified the tumor suppressor, inhibitor of growth 4 (Ing4), as a critical regulator of hematopoietic stem cell (HSC) homeostasis. Cancer cell line models with Ing4 deficiency have shown that Ing4 functions as a tumor suppressor, in part, due to Ing4-mediated regulation of several major signaling pathways, including c-Myc. In HSCs, we show Ing4 deficiency promotes gene expression signatures associated with activation, yet HSCs are arrested in G0, expressing several markers of quiescence. Functionally, Ing4-deficient HSCs demonstrate robust regenerative capacity following transplantation. Our findings suggest Ing4 deficiency promotes a poised state in HSCs, where they appear transcriptionally primed for activation but remain in a resting state. Our model provides key tools for further identification and characterization of pathways that control quiescence and self-renewal in HSCs.

The transcription factor GATA2 has a pivotal role in haematopoiesis. Heterozygous germline GATA2 mutations result in a syndrome characterized by immunodeficiency, bone marrow failure and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukaemia. Clinical symptoms in these patients are diverse and mechanisms driving GATA2-related phenotypes are largely unknown. To explore the impact of GATA2 haploinsufficiency on haematopoiesis, we generated a zebrafish model carrying a heterozygous mutation of gata2b (gata2b+/-), an orthologue of GATA2. Morphological analysis revealed myeloid and erythroid dysplasia in gata2b+/- kidney marrow. Because Gata2b could affect both transcription and chromatin accessibility during lineage differentiation, this was assessed by single-cell (sc) RNA-seq and single-nucleus (sn) ATAC-seq. Sn-ATAC-seq showed that the co-accessibility between the transcription start site (TSS) and a -3.5-4.1 kb putative enhancer was more robust in gata2b+/- zebrafish HSPCs compared to wild type, increasing gata2b expression and resulting in higher genome-wide Gata2b motif use in HSPCs. As a result of increased accessibility of the gata2b locus, gata2b+/- chromatin was also more accessible during lineage differentiation. scRNA-seq data revealed myeloid differentiation defects, that is, impaired cell cycle progression, reduced expression of cebpa and cebpb and increased signatures of ribosome biogenesis. These data also revealed a differentiation delay in erythroid progenitors, aberrant proliferative signatures and down-regulation of Gata1a, a master regulator of erythropoiesis, which worsened with age. These findings suggest that cell-intrinsic compensatory mechanisms, needed to obtain normal levels of Gata2b in heterozygous HSPCs to maintain their integrity, result in aberrant lineage differentiation, thereby representing a critical step in the predisposition to MDS.

During embryonic development the placental vasculature acts as a major hematopoietic niche, where endothelial to hematopoietic transition ensures emergence of hematopoietic stem cells (HSCs). However, the molecular mechanisms that regulate the placental hematoendothelial niche are poorly understood. Using a parietal trophoblast giant cell (TGC)-specific knockout mouse model and single-cell RNA-sequencing, we show that the paracrine factors secreted by the TGCs are critical in the development of this niche. Disruptions in the TGC-specific paracrine signaling leads to the loss of HSC population and the concomitant expansion of a KDR+/DLL4+/PROM1+ hematoendothelial cell-population in the placenta. Combining single-cell transcriptomics and receptor-ligand pair analyses, we also define the parietal TGC-dependent paracrine signaling network and identify Integrin signaling as a fundamental regulator of this process. Our study elucidates novel mechanisms by which non-autonomous signaling from the primary parietal TGCs maintain the delicate placental hematopoietic-angiogenic balance and ensures embryonic and extraembryonic development.

Recent findings suggest that Hematopoietic Stem Cells (HSC) and progenitors arise simultaneously and independently of each other already in the embryonic aorta-gonad mesonephros region, but it is still unknown how their different features are established. Here, we uncover IκBα (Nfkbia, the inhibitor of NF-κB) as a critical regulator of HSC proliferation throughout development. IκBα balances retinoic acid signaling levels together with the epigenetic silencer, PRC2, specifically in HSCs. Loss of IκBα decreases proliferation of HSC and induces a dormancy related gene expression signature instead. Also, IκBα deficient HSCs respond with superior activation to in vitro culture and in serial transplantation. At the molecular level, chromatin regions harboring binding motifs for retinoic acid signaling are hypo-methylated for the PRC2 dependent H3K27me3 mark in IκBα deficient HSCs. Overall, we show that the proliferation index in the developing HSCs is regulated by a IκBα-PRC2 axis, which controls retinoic acid signaling.

The spectrum of myeloid disorders ranges from aplastic bone marrow failure characterized by an empty bone marrow completely lacking in hematopoiesis to acute myeloid leukemia in which the marrow space is replaced by undifferentiated leukemic blasts. Recent advances in the capacity to sequence bulk tumor population as well as at a single-cell level has provided significant insight into the stepwise process of transformation to acute myeloid leukemia. Using models of progression in the context of germ line predisposition (trisomy 21, GATA2 deficiency, and SAMD9/9L syndrome), premalignant states (clonal hematopoiesis and clonal cytopenia of unknown significance), and myelodysplastic syndrome, we review the mechanisms of progression focusing on the hierarchy of clonal mutation and potential roles of transcription factor alterations, splicing factor mutations, and the bone marrow environment in progression to acute myeloid leukemia. Despite major advances in our understanding, preventing the progression of these disorders or treating them at the acute leukemia phase remains a major area of unmet medical need.

Thrombocytopenia 2, an autosomal dominant inherited disease characterized by moderate thrombocytopenia, predisposition to myeloid malignancies and normal platelet size and function, can be caused by 5'-untranslated region (UTR) point mutations in ankyrin repeat domain containing 26 (ANKRD26). Runt related transcription factor 1 (RUNX1) and friend leukemia integration 1 (FLI1) have been identified as negative regulators of ANKRD26. However, the positive regulators of ANKRD26 are still unknown.

To prove the positive regulatory effect of GATA binding protein 2 (GATA2) on ANKRD26 transcription.

Human induced pluripotent stem cells derived from bone marrow (hiPSC-BM) and urothelium (hiPSC-U) were used to examine the ANKRD26 expression pattern in the early stage of differentiation. Then, transcriptome sequencing of these iPSCs and three public transcription factor (TF) databases (Cistrome DB, animal TFDB and ENCODE) were used to identify potential TF candidates for ANKRD26. Furthermore, overexpression and dual-luciferase reporter experiments were used to verify the regulatory effect of the candidate TFs on ANKRD26. Moreover, using the GENT2 platform, we analyzed the relationship between ANKRD26 expression and overall survival in cancer patients.

In hiPSC-BMs and hiPSC-Us, we found that the transcription levels of ANKRD26 varied in the absence of RUNX1 and FLI1. We sequenced hiPSC-BM and hiPSC-U and identified 68 candidate TFs for ANKRD26. Together with three public TF databases, we found that GATA2 was the only candidate gene that could positively regulate ANKRD26. Using dual-luciferase reporter experiments, we showed that GATA2 directly binds to the 5'-UTR of ANKRD26 and promotes its transcription. There are two identified binding sites of GATA2 that are located 2 kb upstream of the TSS of ANKRD26. In addition, we discovered that high ANKRD26 expression is always related to a more favorable prognosis in breast and lung cancer patients.

We first discovered that the transcription factor GATA2 plays a positive role in ANKRD26 transcription and identified its precise binding sites at the promoter region, and we revealed the importance of ANKRD26 in many tissue-derived cancers.

This study investigates the short and long-term effects of IFNT and PAG on the transcriptome of endometrium and blood leukocytes. Holstein heifers received intrauterine infusions of one of the following treatments: 20 mL of a 200 μg/mL bovine serum albumin solution (BSA; vehicle) from day 14 to 16 of the estrous cycle (BSA), vehicle + 10 μg/mL of IFNT from day 14 to 16 (IFNT3), vehicle + 10 μg/mL of IFNT from day 14 to 19 (IFNT6), and vehicle + 10 μg/mL of IFNT from day 14 to 16 followed by vehicle + 10 μg/mL of IFNT + 5 μg/mL of PAG from day 17 to 19 (IFNT+PAG). RNA-seq analysis was performed in endometrial biopsies and blood leukocytes collected after treatments. Acute IFNT signaling in the endometrium (IFNT3 vs BSA), induced differentially expressed genes (DEG) associated with interferon activation, immune response, inflammation, cell death, and inhibited vesicle transport and extracellular matrix remodeling. Prolonged IFNT signaling (IFNT6 vs IFNT3) altered gene expression related to cell invasion, retinoic acid signaling, and embryo implantation. In contrast, PAG induced numerous DEG in blood leukocytes but only 4 DEG in the endometrium. In blood leukocytes, PAG stimulated genes involved in development and TGFB signaling while inhibiting interferon signaling and cell migration. Overall, IFNT is a primary regulator of endometrial gene expression, while PAG predominantly affected the transcriptome of circulating immune cells during early pregnancy. Further research is essential to fully grasp the roles of identified DEG in both the endometrium and blood leukocytes.

During embryogenesis, the fetal liver becomes the main hematopoietic organ, where stem and progenitor cells as well as immature and mature immune cells form an intricate cellular network. Hematopoietic stem cells (HSCs) reside in a specialized niche, which is essential for their proliferation and differentiation. However, the cellular and molecular determinants contributing to this fetal HSC niche remain largely unknown. Macrophages are the first differentiated hematopoietic cells found in the developing liver, where they are important for fetal erythropoiesis by promoting erythrocyte maturation and phagocytosing expelled nuclei. Yet, whether macrophages play a role in fetal hematopoiesis beyond serving as a niche for maturing erythroblasts remains elusive. Here, we investigate the heterogeneity of macrophage populations in the murine fetal liver to define their specific roles during hematopoiesis. Using a single-cell omics approach combined with spatial proteomics and genetic fate-mapping models, we found that fetal liver macrophages cluster into distinct yolk sac-derived subpopulations and that long-term HSCs are interacting preferentially with one of the macrophage subpopulations. Fetal livers lacking macrophages show a delay in erythropoiesis and have an increased number of granulocytes, which can be attributed to transcriptional reprogramming and altered differentiation potential of long-term HSCs. Together, our data provide a detailed map of fetal liver macrophage subpopulations and implicate macrophages as part of the fetal HSC niche.

Mutations in genes encoding transcription factors inactivate or generate ectopic activities to instigate pathogenesis. By disrupting hematopoietic stem/progenitor cells, GATA2 germline variants create a bone marrow failure and leukemia predisposition, GATA2 deficiency syndrome, yet mechanisms underlying the complex phenotypic constellation are unresolved. We used a GATA2-deficient progenitor rescue system to analyze how genetic variation influences GATA2 functions. Pathogenic variants impaired, without abrogating, GATA2-dependent transcriptional regulation. Variants promoted eosinophil and repressed monocytic differentiation without regulating mast cell and erythroid differentiation. While GATA2 and T354M required the DNA-binding C-terminal zinc finger, T354M disproportionately required the N-terminal finger and N terminus. GATA2 and T354M activated a CCAAT/Enhancer Binding Protein-ε (C/EBPε) enhancer, creating a feedforward loop operating with the T-cell Acute Lymphocyte Leukemia-1 (TAL1) transcription factor. Elevating C/EBPε partially normalized hematopoietic defects of GATA2-deficient progenitors. Thus, pathogenic germline variation discriminatively spares or compromises transcription factor attributes, and retaining an obligate enhancer mechanism distorts a multilineage differentiation program.

For almost two decades, clinicians have overlooked the diagnostic potential of CD34neg hematopoietic stem cells because of their limited homing capacity relative to CD34posHSCs when injected intravenously. This has contributed to the lack of appeal of using umbilical cord blood in HSC transplantation because its stem cell count is lower than bone marrow. The present study reveals that the homing and engraftment of CD34negHSCs can be improved by adding the Sialyl Lewis X molecule via α1,3-fucosylation. This unlocks the potential for using this more primitive stem cell to treat blood disorders because our findings show CD34negHSCs have the capacity to regenerate cells in the bone marrow of mice for several months. Furthermore, our RNA sequencing analysis revealed that CD34negHSCs have unique adhesion pathways, downregulated in CD34posHSCs, that facilitate interaction with the bone marrow niche. Our findings suggest that CD34neg cells will best thrive when the HSC resides in its microenvironment.

The Polycomb Repressive Complex 2 (PRC2) methylates H3K27 to regulate development and cell fate by transcriptional silencing. Alteration of PRC2 is associated with various cancers. Here, we show that mouse Kdm1a deletion causes a dramatic reduction of PRC2 proteins, whereas mouse null mutation of L3mbtl3 or Dcaf5 results in PRC2 accumulation and increased H3K27 trimethylation. The catalytic subunit of PRC2, EZH2, is methylated at lysine 20 (K20), promoting EZH2 proteolysis by L3MBTL3 and the CLR4DCAF5 ubiquitin ligase. KDM1A (LSD1) demethylates the methylated K20 to stabilize EZH2. K20 methylation is inhibited by AKT-mediated phosphorylation of serine 21 in EZH2. Mouse Ezh2K20R/K20R mutants develop hepatosplenomegaly associated with high GFI1B expression, and Ezh2K20R/K20R mutant bone marrows expand hematopoietic stem cells and downstream hematopoietic populations. Our studies reveal that EZH2 is regulated by methylation-dependent proteolysis, which is negatively controlled by AKT-mediated S21 phosphorylation to establish a methylation-phosphorylation switch to regulate the PRC2 activity and hematopoiesis.

GATA binding protein 2 (GATA2) is a conserved zinc finger transcription factor that regulates the emergence and maintenance of complex genetic programs driving development and function of hematopoietic stem and progenitor cells (HSPCs). Patients born with monoallelic GATA2 mutations develop myelodysplastic neoplasm (MDS) and acute myeloid leukemia (AML), whereas acquired GATA2 mutations are reported in 3% to 5% of sporadic AML cases. The mechanisms by which aberrant GATA2 activity promotes MDS and AML are incompletely understood. Efforts to understand GATA2 in basic biology and disease will be facilitated by the development of broadly efficacious antibodies recognizing physiologic levels of GATA2 in diverse tissue types and assays. Here, we purified a polyclonal anti-GATA2 antibody and generated multiple highly specific anti-GATA2 monoclonal antibodies, optimized them for immunohistochemistry on patient bone marrow bioosy samples, and analyzed GATA2 expression in adults with healthy bone marrow, MDS, and acute leukemia. In healthy bone marrow, GATA2 was detected in mast cells, subsets of CD34+ HSPCs, E-cadherin-positive erythroid progenitors, and megakaryocytes. In MDS, GATA2 expression correlates with bone marrow blast percentage, positively correlates with myeloid dysplasia and complex cytogenetics, and is a nonindependent negative predictor of overall survival. In acute leukemia, the percent of GATA2+ blasts closely associates with myeloid lineage, whereas a subset of lymphoblastic and undifferentiated leukemias with myeloid features also express GATA2. However, the percent of GATA2+ blasts in AML is highly variable. Elevated GATA2 expression in AML blasts correlates with peripheral neutropenia and complex AML cytogenetics but, unlike in MDS, does not predict survival.

Bladder cancer (BLCA) is a commonly diagnosed cancer worldwide that exhibits high rates of recurrence and metastasis. Immunotherapy is increasingly being recognised in the clinical management of bladder cancer. In addition, the prospect of developing Natural Killer (NK) cell-related immunotherapy is promising in BLCA.

We established and verified a prognostic signature based on NK cell-related gene expression. We then calculated the NKscore of BLCA samples and correlated it with the clinical outcomes, molecular subtypes of BLCA, tumour microenvironment (TME), and predicted efficacy of immune checkpoint inhibitors (ICI) and chemotherapy drugs to thoroughly explore the implications of the NKscore. Finally, the role of the NK signature gene HECTD1 in BLCA was verified by Quantitative Real-time PCR, Cell Counting Kit-8 Assay (CCK-8), Transwell Assay and Colony Formation Experiment.

We analysed NK cell-associated genes and identified six genes with significant prognostic relevance. A high NK score significantly represents a worse prognosis. NKscore was significantly correlated with seven types of classical molecular subtype classifications of BLCA. In addition, NKscore positively correlates with NK-related immune checkpoints, suggesting that emerging NK cell immune checkpoint inhibitors, such as monalizumab, may have potential therapeutic promise for patients with high NKscore. The results of the T cell inflamed score (TIS) and tumour immune dysfunction exclusion (TIDE) score confirmed the suitability of immunotherapy for patients with a high NK score. Likewise, patients with a high NK score may be more suitable for several significant chemotherapeutic drugs. Functional experiments showed that the knockdown of HECTD1 significantly attenuated the proliferation, migration, and invasion ability of tumour cells.

To sum up, the capability of our signature to predict prognosis and immunotherapy response was robust. Hopefully, these results will provide new insights for BLCA research and patient immunotherapy.

Transcription factors (TFs) play a crucial role in regulating the dynamic and precise patterns of gene expression required for the initial specification of endothelial cells (ECs), and during endothelial growth and differentiation. While sharing many core features, ECs can be highly heterogeneous. Differential gene expression between ECs is essential to pattern the hierarchical vascular network into arteries, veins and capillaries, to drive angiogenic growth of new vessels, and to direct specialization in response to local signals. Unlike many other cell types, ECs have no single master regulator, instead relying on differing combinations of a necessarily limited repertoire of TFs to achieve tight spatial and temporal activation and repression of gene expression. Here, we will discuss the cohort of TFs known to be involved in directing gene expression during different stages of mammalian vasculogenesis and angiogenesis, with a primary focus on development.

Regenerative medicine is a tool to compensate for the shortage of lungs for transplantation, but it remains difficult to construct a lung in vitro due to the complex three-dimensional structures and multiple cell types required. A blastocyst complementation method using interspecies chimeric animals has been attracting attention as a way to create complex organs in animals, although successful lung formation using interspecies chimeric animals has not yet been achieved. Here, we applied a reverse-blastocyst complementation method to clarify the conditions required to form lungs in an Fgfr2b-deficient mouse model. We then successfully formed a rat-derived lung in the mouse model by applying a tetraploid-based organ-complementation method. Importantly, rat lung epithelial cells retained their developmental timing even in the mouse body. These findings provide useful insights to overcome the barrier of species-specific developmental timing to generate functional lungs in interspecies chimeras.

Germline genetic variants alter the coding and enhancer sequences of GATA2, which encodes a master regulator of hematopoiesis. The conserved murine Gata2 enhancer (+9.5) promotes hematopoietic stem cell (HSC) genesis during embryogenesis. Heterozygosity for a single-nucleotide Ets motif variant in the human enhancer creates a bone marrow failure and acute myeloid leukemia predisposition termed GATA2 deficiency syndrome. The homozygous murine variant attenuates chemotherapy- and transplantation-induced hematopoietic regeneration, hematopoietic stem and progenitor cell (HSPC) response to inflammation, and HSPC mobilization with the therapeutic mobilizer granulocyte colony-stimulating factor (G-CSF). Because a Gata2 +9.5 variant attenuated G-CSF-induced HSPC expansion and mobilization, and HSC transplantation therapies require efficacious mobilization, we tested whether variation affects mechanistically distinct mobilizers or only those operating through select pathways. In addition to affecting G-CSF activity, Gata2 variation compromised IL-8/CXCR2- and VLA-4/VCAM1-induced mobilization. Although the variation did not disrupt HSPC mobilization mediated by plerixafor, which functions through CXCR4/CXCL12, homozygous and heterozygous variation attenuated mobilization efficacy of the clinically used plerixafor/G-CSF combination. The influence of noncoding variation on HSPC mobilization efficacy and function is important clinically because comprehensive noncoding variation is not commonly analyzed in patients. Furthermore, our mobilization-defective system offers unique utility for elucidating fundamental HSPC mechanisms.

The bone marrow is responsible for producing an incredible number of cells daily in order to maintain blood homeostasis through a process called hematopoiesis. Hematopoiesis is a greatly demanding process and one entirely dependent on complex interactions between the hematopoietic stem cell (HSC) and its surrounding microenvironment. Zinc (Zn2+) is considered an important trace element, playing diverse roles in different tissues and cell types, and zinc finger proteins (ZNF) are proteins that use Zn2+ as a structural cofactor. In this way, the ZNF structure is supported by a Zn2+ that coordinates many possible combinations of cysteine and histidine, with the most common ZNF being of the Cys2His2 (C2H2) type, which forms a family of transcriptional activators that play an important role in different cellular processes such as development, differentiation, and suppression, all of these being essential processes for an adequate hematopoiesis. This review aims to shed light on the relationship between ZNF and the regulation of the hematopoietic tissue. We include works with different designs, including both in vitro and in vivo studies, detailing how ZNF might regulate hematopoiesis.

The correct maintenance and differentiation of hematopoietic stem cells (HSC) in bone marrow is vital for the maintenance and operation of the human blood system. GATA2 plays a critical role in the maintenance of HSCs and the specification of HSCs into the different hematopoietic lineages, highlighted by the various defects observed in patients with heterozygous mutations in GATA2, resulting in cytopenias, bone marrow failure and increased chance of myeloid malignancy, termed GATA2 deficiency syndrome. Despite this, the mechanisms underlying GATA2 deficiency syndrome remain to be elucidated. The detailed description of how GATA2 regulates HSC maintenance and blood lineage determination is crucial to unravel the pathogenesis of GATA2 deficiency syndrome. In this review, we summarize current advances in elucidating the role of GATA2 in hematopoietic cell fate determination and discuss the challenges of modeling GATA2 deficiency syndrome.

Blood comprises a wide array of specialized cells, all of which share the same genetic information and ultimately derive from the same precursor, the hematopoietic stem cell (HSC). This diversity of phenotypes is underpinned by unique transcriptional programs gradually acquired in the process known as hematopoiesis. Spatiotemporal regulation of gene expression depends on many factors, but critical among them are enhancers-sequences of DNA that bind transcription factors and increase transcription of genes under their control. Thus, hematopoiesis involves the activation of specific enhancer repertoires in HSCs and their progeny, driving the expression of sets of genes that collectively determine morphology and function. Disruption of this tightly regulated process can have catastrophic consequences: in hematopoietic malignancies, dysregulation of transcriptional control by enhancers leads to misexpression of oncogenes that ultimately drive transformation. This review attempts to provide a basic understanding of enhancers and their role in transcriptional regulation, with a focus on normal and malignant hematopoiesis. We present examples of enhancers controlling master regulators of hematopoiesis and discuss the main mechanisms leading to enhancer dysregulation in leukemia and lymphoma.

The transcription factor GATA2 is involved in human diseases ranging from hematopoietic disorders, to cancer, to infectious diseases. GATA2 is one of six GATA-family transcription factors that act as pioneering transcription factors which facilitate the opening of heterochromatin and the subsequent binding of other transcription factors to induce gene expression from previously inaccessible regions of the genome. Although GATA2 is essential for hematopoiesis and lymphangiogenesis, it is also expressed in other tissues such as the lung, prostate gland, gastrointestinal tract, central nervous system, placenta, fetal liver, and fetal heart. Gene or transcriptional abnormalities of GATA2 causes or predisposes patients to several diseases including the hematological cancers acute myeloid leukemia and acute lymphoblastic leukemia, the primary immunodeficiency MonoMAC syndrome, and to cancers of the lung, prostate, uterus, kidney, breast, gastric tract, and ovaries. Recent data has also linked GATA2 expression and mutations to responses to infectious diseases including SARS-CoV-2 and Pneumocystis carinii pneumonia, and to inflammatory disorders such as atherosclerosis. In this article we review the role of GATA2 in the etiology and progression of these various diseases.

In mitosis, most transcription factors detach from chromatin, but some are retained and bookmark genomic sites. Mitotic bookmarking has been implicated in lineage inheritance, pluripotency and reprogramming. However, the biological significance of this mechanism in vivo remains unclear. Here, we address mitotic retention of the hemogenic factors GATA2, GFI1B and FOS during haematopoietic specification. We show that GATA2 remains bound to chromatin throughout mitosis, in contrast to GFI1B and FOS, via C-terminal zinc finger-mediated DNA binding. GATA2 bookmarks a subset of its interphase targets that are co-enriched for RUNX1 and other regulators of definitive haematopoiesis. Remarkably, homozygous mice harbouring the cyclin B1 mitosis degradation domain upstream Gata2 partially phenocopy knockout mice. Degradation of GATA2 at mitotic exit abolishes definitive haematopoiesis at aorta-gonad-mesonephros, placenta and foetal liver, but does not impair yolk sac haematopoiesis. Our findings implicate GATA2-mediated mitotic bookmarking as critical for definitive haematopoiesis and highlight a dependency on bookmarkers for lineage commitment.

ST6GALNAC family members function as sialyltransferases and have been implicated in cancer progression. However, their aberrant expression levels, prognostic values and specific roles in metastatic prostate cancer (PCa) remain largely unclear.

Two independent public datasets (TCGA-PRAD and GSE21032), containing 648 PCa samples in total, were employed to comprehensively examine the mRNA expression changes of ST6GALNAC family members in PCa, as well as their associations with clinicopathological parameters and prognosis. The dysregulation of ST6GALNAC5 was further validated in a mouse PCa model and human PCa samples from our cohort (n = 64) by immunohistochemistry (IHC). Gene Set Enrichment Analysis, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes and drug sensitivity analyses were performed to enrich the biological processes most related to ST6GALNAC5. Sulforhodamine B, transwell, luciferase reporter and chromatin immunoprecipitation (ChIP) assays were used to examine the PCa cell proliferation, invasion and transcriptional regulation, respectively.

Systematical investigation of six ST6GALNAC family members in public datasets revealed that ST6GALNAC5 was the only gene consistently and significantly upregulated in metastatic PCa, and ST6GALNAC5 overexpression was also positively associated with Gleason score and predicted poor prognosis in PCa patients. IHC results showed that (1) ST6GALNAC5 protein expression was increased in prostatic intraepithelial neoplasia and further elevated in PCa from a PbCre;Pten^F/F mouse model; (2) overexpressed ST6GALNAC5 protein was confirmed in human PCa samples comparing with benign prostatic hyperplasia samples from our cohort (p < 0.001); (3) ST6GALNAC5 overexpression was significantly correlated with perineural invasion of PCa. Moreover, we first found transcription factor GATA2 positively and directly regulated ST6GALNAC5 expression at transcriptional level. ST6GALNAC5 overexpression could partially reverse GATA2-depletion-induced inhibition of PCa cell invasion. The GATA2-ST6GALNAC5 signature exhibited better prediction on the poor prognosis in PCa patients than GATA2 or ST6GALNAC5 alone.

Our results indicated that GATA2-upregulated ST6GALNAC5 might serve as an adverse prognostic biomarker promoting prostate cancer cell invasion.